Agent for ameliorating pancreatic function disorder

ABSTRACT

Agents for protecting or ameliorating pancreatic cells and tissues which contains as the active ingredient a neurotrophic factor such as BDNF. By using these drugs, degenerative dropout of pancreatic cells and pancreatic hypofunction caused by diabetes, acute/chronic pancreatitis, etc. can be efficaciously prevented and treated.

This application is the national phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/JP00/02264 which has an Internationalfiling date of Apr. 7, 2000, which designated the United States ofAmerica and was not published in English.

TECHNICAL FIELD

The present invention relates to an agent for protecting pancreaticcells or an agent for ameliorating hypofunction of pancreatic cells, oran agent for protecting pancreatic tissues or an agent for amelioratinghypofunction of pancreatic tissues, these agent comprising as the activeingredient a neurotrophic factor.

BACKGROUND ART

Pancreas is an organ consisting of the endocrine gland tissues calledpancreatic islet (Langerhans islet) and the exocrine gland tissuessecreting digestive enzymes such as amylase, lipase, protease, etc. Inthe Langerhans islet, B cells (β cells) synthesizing and secretinginsulin, etc., A cells (α cell) synthesizing and secreting glucagon,etc., D cells (δ cells) synthesizing and secreting somatostatin, andpancreatic polypeptide cells (hereinafter, referred to as PP cells)synthesizing and secreting pancreatic polypeptide, etc. exist, and theygreatly affect the control of blood glucose and metabolism. Disorders ofthese endocrine glands and exocrine glands may induce abnormalities ofcontrolling blood glucose level (e.g., diabetes mellitus, hypoglycemia,insulin shock, etc.) and decreased digestion (e.g., steatorrhea, etc.),respectively.

Pancreatic function disorders are induced by various causes, andrepresentative underlying diseases thereof are, for example,pancreatitis or diabetes mellitus. Pancreatitis is clinically classifiedinto acute pancreatitis and chronic pancreatitis, and the former ismono-pancreatitis or repetitive pancreatitis being characterized byacute bellyache attack accompanied by increase in pancreatic enzymelevel in blood or urine. Serious acute pancreatitis further inducesnecrosis and hemorrhage of pancreatic substratum, serious renal failure,or respiratory failure, and may results in shock to death. The treatmentthereof is usually carried out by inhibiting pancreatic exocrine byfasting and an H2 blocker, and further by preventing complications byadministering a protease inhibitor, an antibiotic, or an analgesic.

Chronic pancreatitis is mainly induced by over-uptake of alcohol, andcharacterized by repetitive or persistent bellyache. Morphologically, itis characterized by immethodical sclerosis accompanied by destructionand permanent dissipation of pancreatic exocrine tissues, and it inducessymptoms caused by pancreatic exocrine grand failure such assteatorrhea. In chronic pancreatitis, it is observed that about 50% ofthe patients produce a complication of diabetes mellitus due topancreatic endocrine disorder (pancreatic diabetes). The characteristicof the secondary diabetes of this chronic pancreatitis is the lack ofboth insulin and glucagon, and the treatment thereof is mostly carriedout by administration of insulin. About half of the causes of death forchronic pancreatitis are concerned with diabetes mellitus, andhypoglycemia after insulin injection (i.e., insulin shock) caused by thelack of glucagon or diabetic complications such as nephropathy orinfections are pointed out as a cause of death.

In addition, sulfonylurea derivatives having an insulin secretionpromoting activity have been used in the treatment of diabetes mellitus,but they may occasionally induce pancreatic cell dysfunction orpancreatic tissue dysfunction, due to excessive burden on the pancreasby forcing the pancreas to secrete insulin.

At present, a method for promoting a spontaneous recovery of pancreaticfunction has been used in the treatment of pancreatic function disorder,by eliminating diseases or factors that are a cause therefor (cf.,“Learning of Pancreatopathy”, edited by Tadashi TAKEUCHI, published byNankodo Co. Ltd., Aug. 1, 1993), but there in have not been known orused any method or agent for aggressively recovering the decreasedpancreatic function.

On the other hand, neurotrophic factors are a generic name for proteins,which are provided from target cells or neurons and glia cells andSchwann cells in the living body. They show activities of maintainingthe survival and differentiation of neurons, and are classified intomany types according to the kinds of nerves or receptors to function.Among them, proteins being known as neurotrophins have high structuralhomology with each other and form a family. The typical examples thereofare neurotrophins such as nerve growth factor (hereinafter, abbreviatedas NGF), brain-derived neurotrophic factor (hereinafter, abbreviated asBDNF), neurotrophin 3 (hereinafter, abbreviated as NT-3), neurotrophin 4(hereinafter, abbreviated as NT-4), neurotrophin 5 (hereinafter,abbreviated as NT-5), or neurotrophin 6 (NT-6); ciliary neurotrophicfactor (hereinafter, abbreviated as CNTF); glia cell-derivedneurotrophic factor (hereinafter, abbreviated as GDNF), etc. Inaddition, neurotrophins are known to act as a specific ligand ofreceptors (trkA, trkB and/or trkC), which are the products of p-75 andtrk genes (cf. Takeshi NONOMURA, Hiroshi HATANAKA; Jikken Igaku, vol.13, p. 376 (1995)).

Neurotrophic factors have been studied with respect to their medical useas a therapeutic agent for treating a patient of neurodegenerativediseases. For example, Society for Neuroscience, vol. 21, p. 1535(1995), A. P. Mizisin et al. discloses the pharmacological activity ofBDNF on diabetic peripheral neuropathy, but this literature merelysuggests the possible pharmacological activity of BDNF on neuropathybased on the finding that BDNF improves the reduction of motor nerveconduction in vivo. WO 98/32458 discloses that neurotrophic factors suchas BDNF can normalize the blood glucose level of diabetic animal models,and applications thereof onto the treatment of diabetes mellitus aredisclosed therein.

DISCLOSURE OF INVENTION

As mentioned above, an agent for protecting pancreatic cells or an agentfor ameliorating damaged pancreatic cells, an agent for protectingpancreatic cell function or an agent for ameliorating pancreatic cellhypofunction, or an agent for protecting pancreatic tissues or an agentfor ameliorating damaged pancreatic tissues, or an agent for protectingpancreatic tissue function or an agent for ameliorating pancreatictissue hypofunction has been desired in the medical field.

The present inventors have an interest in that the insulin secretion ofBDNF-treated type 2 diabetic animal models is kept at high level, andhave studied pancreatic function ameliorating activities by using type 2diabetic animal models. As a result, they have found that BDNF can (1)increase the decreased insulin content in pancreas of type 2 diabetesanimal models, (2) reduce the increased glucagon content in pancreas,(3) normalize the localization of A cells and D cells in the pancreaticLangerhans islet, (4) promote the re-granulation of insulin secretorygranules of B cells in the pancreatic Langerhans islet, and normalizethe organellae. Based on the finding of these pancreatic functionameliorating activity and pancreatic cell protecting activity of BDNF,the present inventors have further studied and have accomplished thepresent invention.

More particular, the present invention relates to the following:

1. An agent for protecting pancreatic cells or an agent for amelioratingdamaged pancreatic cells, which comprises as the active ingredient aneurotrophic factor;

2. An agent for protecting pancreatic cell function or an agent forameliorating pancreatic cell hypofunction, which comprises as the activeingredient a neurotrophic factor;

3. An agent for protecting pancreatic tissues or an agent forameliorating damaged pancreatic tissues, which comprises as the activeingredient a neurotrophic factor;

4. An agent for protecting pancreatic tissue function or an agent forameliorating pancreatic tissue hypofunction, which comprises as theactive ingredient a neurotrophic factor;

5. The agent for protection or amelioration of the function according tothe above 1 or 2, wherein the pancreatic cell is B cells (β cells), Acells (α cells), and/or D cells (δ cells) of the pancreatic Langerhansislet;

6. The agent for protection or amelioration of the function according tothe above 1 or 2, wherein the pancreatic cell is B cells (β cells), Acells (α cells), D cells (δ cells), and/or PP cells of the pancreaticLangerhans islet;

7. The agent for protecting pancreatic tissue function or the agent forameliorating hypofunction of pancreatic tissue according to the above 3or 4, wherein the pancreatic tissue is the pancreatic Langerhans islet;

8. The agent for protection or amelioration according to the above 1, 2,5 or 6, which is an agent for protecting or ameliorating pancreaticendocrine function (insulin secretion ability, glucagon secretionability and/or somatostatin secretion ability) or an agent forameliorating pancreatic endocrine function disorder (insulin secretionability, glucagon secretion ability and/or somatostatin secretionability);

9. The agent for protection or amelioration according to the above 3, 4or 7, which is an agent for protecting or ameliorating pancreaticendocrine function (insulin secretion ability, glucagon secretionability and/or somatostatin secretion ability) or an agent forameliorating pancreatic endocrine function disorder (insulin secretionability, glucagon secretion ability and/or somatostatin secretionability);

10. The agent for protection or amelioration according to the above 1,2, 5, 6, or 8, wherein the cause for hypofunction or disorder ofpancreatic cells is diabetes mellitus;

11. The agent for protection or amelioration according to the above 3,4, 7 or 9, wherein the cause for hypofunction or disorder of pancreatictissues is diabetes mellitus;

12. The agent for protection or amelioration according to the above 1,2, 5, 6, or 8, wherein the cause for hypofunction or disorder ofpancreatic cells is acute pancreatitis or chronic pancreatitis;

13. The agent for protection or amelioration according to the above 3,4, 7 or 9, wherein the cause for hypofunction or disorder of pancreatictissues is acute pancreatitis or chronic pancreatitis;

14. The agent for protection or amelioration according to the above 1,2, 5, 6, or 8, wherein the cause for hypofunction or disorder ofpancreatic cells is a sulfonylurea derivative;

15. The agent for protection or amelioration according to the above 3,4, 7 or 9, wherein the cause for hypofunction or disorder of pancreatictissues is a sulfonylurea derivative;

16. An agent for treating acute pancreatitis or chronic pancreatitis,which comprises as the active ingredient the agent for protection oramelioration as set forth in any one of the above 1, 2, 5, 6, and 8;

17. An agent for treating acute pancreatitis or chronic pancreatitis,which comprises as the active ingredient the agent for protection oramelioration as set forth in any one of the above 3, 4, 7 and 9;

16. An agent for protecting pancreatic endocrine tissues or an agent forameliorating damaged pancreatic endocrine tissues, which comprises asthe active ingredient a neurotrophic factor;

17. An agent for protecting pancreatic endocrine tissue function or anagent for ameliorating hypofunction of pancreatic endocrine tissues,which comprises as the active ingredient a neurotrophic factor;

18. An agent for protecting pancreatic exocrine tissues or an agent forameliorating damaged pancreatic exocrine tissues, which comprises as theactive ingredient a neurotrophic factor;

19. An agent for protecting pancreatic exocrine tissue function or anagent for ameliorating hypofunction of pancreatic exocrine tissues,which comprises as the active ingredient a neurotrophic factor;

20. The agent for protection or amelioration according to any one of theabove 1 to 19, wherein the neurotrophic factor of the active ingredientis NGF (nerve growth factor), BDNF (brain-derived neurotrophic factor),CNTF (ciliary neurotrophic factor), NT-3 (neurotrophin 3), NT-4(neurotrophin 4), NT-5 (neurotrophin 5), or NT-6 (neurotrophin 6);

21. The agent for protection or amelioration according to any one of theabove 1 to 19, wherein the neurotrophic factor of the active ingredientis BDNF (brain-derived neurotrophic factor);

22. The agent for protection or amelioration according to any one of theabove 1 to 19, wherein the neurotrophic factor of the active ingredientis CNTF (ciliary neurotrophic factor);

23. The agent for protection or amelioration according to any one of theabove 1 to 19, wherein the neurotrophic factor of the active ingredientis NT-3 (neurotrophin 3);

24. The agent for protection or amelioration according to any one of theabove 1 to 19, wherein the neurotrophic factor of the active ingredientis NT-4 (neurotrophin 4);

25. The agent for protection or amelioration according to any one of theabove 1 to 19, wherein the neurotrophic factor of the active ingredientis NT-5 (neurotrophin 5);

26. The agent for protection or amelioration according to any one of theabove 1 to 19, wherein the neurotrophic factor of the active ingredientis NT-6 (neurotrophin 6);

27. The agent for protection or amelioration according to any one of theabove 1 to 19, wherein the neurotrophic factor of the active ingredientis NGF (nerve growth factor);

28. The agent for protection or amelioration according to any one of theabove 1 to 19, wherein the neurotrophic factor of the active ingredientis GDNF (glia cell-derived neurotrophic factor);

29. The agent for protection or amelioration according to any one of theabove 1 to 19, wherein the neurotrophic factor of the active ingredientis a trkA, a trkB and/or a trkC receptor agonist;

The meaning or definition of each term used in the present specificationis explained below.

The “agent for protection” means an agent for preventing disorder orhypofunction.

The “agent for amelioration” means an agent for adjusting a damagedcondition or a condition of hypofunction to the normal condition ornormalizing it.

The “neurotrophic factor” means a physiologically active substance,which is secreted from the target cells for nerve growth, or byautocrine or paracrine, and promotes the growth, differentiation, orsurvival of neurons to form a neural circuit (synapse) in the livingbody. For example, the neurotrophic factor includes neurotrophins suchas a nerve growth factor (hereinafter, abbreviated as NGF), abrain-derived neurotrophic factor (hereinafter, abbreviated as BDNF), aneurotrophin 3 (hereinafter, abbreviated as NT-3), a neurotrophin 4(hereinafter, abbreviated as NT-4), a neurotrophin 5 (hereinafter,abbreviated as NT-5), and a neurotrophin 6 (hereinafter, abbreviated asNT-6); ciliary neurotrophic factor (hereinafter, abbreviated as CNTF);glia cell-derived neurotrophic factor (hereinafter, abbreviated asGDNF), etc. In addition, a modified recombinant neurotrophic factorproduced by a substitution, a deletion, or an addition of a part ofamino acid sequence of the naturally occurred neurotrophic factorsequence by a conventional technique may be included in the neurotrophicfactor of the present specification, as far as it exhibits the similarphysiological activity.

The “pancreatic function disorder” means pathologies, wherein theendocrine gland function or exocrine gland function of the pancreas isdecreased or abnormally elevated. The endocrine gland function mainlymeans the secretion ability of insulin, glucagon and/or somatostatin,and the exocrine gland function mainly means the secretion ability ofdigestive enzymes (amylase, protease and/or lipase) into pancreaticjuice. These secretion abilities can be evaluated by a conventionalmethod being widely used in the clinical field, for example by measuringblood insulin level.

The “protection of pancreatic cells” means an action of protectingpancreatic endocrine gland cells and exocrine gland cells fromdegeneration induced by various causes, and it can be evaluated byexamination of pancreatic tissue section under a microscope. Endocrinegland cells can specifically be stained by a conventional cell stainingsuch as aldehyde fuchsin stain, and in the pancreatic Langerhans islet,B cells (β cells) can be stained by insulin immune staining, and A cells(α cells) can be stained by glucagon immune staining, and D cells (δcells) can be stained by somatostatin immune staining, and the stainedpatterns thereof can be observed. In addition, the structure oforganellae can be examined by electron microscopy for evaluation.

The “protection of pancreatic tissues” means an action of protectingpancreatic endocrine gland tissues and exocrine gland tissues fromdegeneration induced by various causes, and it can be evaluated byexamination of pancreatic tissue section under a microscope. The stainedpattern of the tissues can be examined by a conventional cell strainingsuch as hematoxylin-eosin stain, aldehyde fuchsin stain, etc. Inaddition, the structure of organellae can be examined by electronmicroscopy for evaluation.

The “trkA, trkB and/or trkC receptor agonist” is a generic name forsubstances that are bound to trkA, trkB or trkC, which is among the trkgene expression products being known as receptors for “neurotrophin”,and activate them to exhibit their activities. Concretely, the knownneurotrophin is, for example, NGF binding to trkA, BDNF and NT-4 bindingto trkB, and NT-3 binding to trkC, etc. This concept includes not onlymodified neurotrophins (modified by amino acid substitution, deletion oraddition or sugar chain modification), but also peptides and organiccompounds of a lower molecular weight as far as they exhibit a bindingability and activating ability to trkA, trkB or trkC receptor, forexample, phosphorilating activity of tyrosine residue.

The “sulfonylurea derivative” is, for example, tolbutamide,acetohexamide, chlorpropamide, glyclopyramide, tolazamide, gliclazide,glibenclamide, glybuzole, glymidine, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photomicrograph by optical microscope of the stained tissueof pancreas. A: the stained picture by aldehyde fuchsin, B: the stainedpicture by insulin immune staining, C: the stained picture by glucagonimmune staining, and D: the stained picture of somatostatin immunestaining. The db/db (vehicle) means diabetic control mice, the db/db(BDNF) means diabetic BDNF-treated mice, and the db/m (normal) meansnormal control mice.

FIG. 2 is an electron micrography (magnification: 8300) of pancreatic Bcells of diabetic control mice.

FIG. 3 is an electron micrography (magnification: 8300) of pancreatic Bcells of BDNF-treated mice.

FIG. 4 is an electron micrography (magnification: 8300) of pancreatic Bcells of normal mice.

FIG. 5 is an electron micrography (magnification: 16000) of pancreatic Bcells of diabetic control mice.

FIG. 6 is an electron micrography (magnification: 16000) of pancreatic Bcells of BDNF-treated mice.

FIG. 7 is an electron micrography (magnification: 16000) of pancreatic Bcells of normal mice.

BEST MODE FOR CARRYING OUT THE INVENTION

The neurotrophic factors used as an active ingredient of the presentinvention may be commercially available ones or can be prepared by thefollowing methods.

The neurotrophic factors used as the active ingredient of the presentinvention can be any ones prepared by various methods as far as they arepurified to such a degree that it could be used as a medicament. Theneurotrophic factor can be obtained by cultivating a primary culturecell or an established cell line that can produce the neurotrophicfactor, and isolating and purifying it from the culture medium thereof(e.g., culture supernatant, cultured cells). Moreover, a recombinantneurotrophic factor can be obtained by a conventional gene engineeringtechnique, e.g., by inserting a gene encoding a neurotrophic factor intoa suitable vector, transforming a suitable host with the recombinantvector, and isolating from a culture supernatant of the resultingtransformant. The host cells to be used in the above process are notlimited, and may be any conventional host cells which have been used ina gene engineering technique, for example, Escherichia coli, Bacillussubtilis, yeasts, mold fungi, plant cells or animal cells.

The neurotrophic factors obtained in the above method include a modifiedrecombinant neurotrophic factor such as ones produced by a deletion of apart of amino acid sequence, or a substitution by other amino acid(s),or an addition of a part of other amino acid sequence, or ones havingone or more amino acids at the N-terminus and/or the C-terminus, or oneswherein the sugar chain is deleted or substituted, as far as theyexhibit substantially the same activity.

Method for Preparation of BDNF

When a conventional gene engineering technique is employed, BDNF isprepared by inserting a gene encoding BDNF into a suitable vector,transforming a suitable host with the recombinant vector, and isolatingit from a culture supernatant of the resulting transformant (cf., Proc.Natl. Acad. Sci. USA, vol. 88, p. 961 (1991); Biochem. Biophys. Res.Commun., vol. 186, p. 1553 (1992)). The gene engineering technique issuitable for production of BDNF of same quality in a large scale. Thehost cells mentioned above are not limited, but may be any conventionalhost cells which have been used in a gene engineering technique, forexample, Escherichia coli, Bacillus subtilis, yeasts, plant cells oranimal cells.

Method for Preparation of NT-3

NT-3 can be prepared by expressing in various host cells in the samemanner as in the preparation of BDNF. The methods for preparing thereofand the methods for assay thereof are disclosed in Neuron, vol. 4,767-773 (1990), or JP-A-5-161493 (WO 91/3659).

Method for Preparation of NT-4

NT-4 can be prepared by expressing in various host cells in the samemanner as in the preparation of BDNF. The methods for expression of therecombinant NT-4 and the methods for assay thereof are disclosed inProc. Natl. Acad. Sci. USA, vol. 89, p. 3060-3064 (1992.4),JP-A-7-509600 (WO 93/25684), or JP-A-6-501617 (WO 92/5254).

Method for Preparation of CNTF

CNTF can be prepared in a large scale by expressing in various hostcells in the same manner as in the preparation of BDNF. The methods forexpression of the recombinant CNTF and the methods for assay thereof aredisclosed in Biochimica et Biophysica Acta, vol. 1090, p. 70-80 (1991),J. Neurochemistry, vol. 57, p. 1003-1012 (1991). The methods forpreparing the recombinant CNTF and the purification thereof aredisclosed in JP-A-4-502916 (WO 90/7341).

The agent for protecting or ameliorating pancreatic cell function, or anagent for protecting or ameliorating pancreatic tissue function, whichcomprise as the active ingredient a neurotrophic factor, can beadministered either parenterally or orally.

The precise dosage and the administration schedule of the above agentsof the present invention should vary according to the dosage to berequired for each patient, the method for treatment, the disease to betreated, or the degree of necessity, and further according to thediagnosis by a physician. When administered parenterally, the dosage andthe frequency of the administration may vary according to theconditions, ages, body weights of patients, and administration routes,but when it is administered subcutaneously or intravenously in the formof an injection, then the daily dosage thereof is in the range of about1 to about 2500 μg, preferably in the range of about 10 to about 500 μgper 1 kg of the body weight in an adult. When it is administered to theair tract in the form of an aerosol spray, the daily dosage thereof isin the range of about 1 μg to about 2500 μg, preferably in the range ofabout 10 to about 500 μg per 1 kg of the body weight in an adult. Theadministration schedule is either continuous daily administration,intermittent administration, or a schedule of combining these methods.

When administered orally, the dosage and the frequency of administrationmay vary according to the conditions, ages, body weights of patients,and administration routes, and the daily dosage thereof is in the rangeof about 5 to about 2500 μg, preferably in the range of about 10 toabout 1000 μg per 1 kg of the body weight in an adult.

A pharmaceutical composition can be prepared by mixing a neurotrophicfactor with a pharmaceutically acceptable non-toxic carrier. When apharmaceutical composition for parenteral administration (subcutaneousinjection, intramuscular injection, or intravenous injection) isprepared, it is preferably in the form of a solution preparation or asuspension preparation. When a pharmaceutical composition forintravaginal administration or rectal administration is prepared, it ispreferably in the form of a semi-solid preparation such as cream orsuppository. When a pharmaceutical composition for intranasaladministration is prepared, it is preferably in the form of a powder, anasal drop, or an aerosol.

The pharmaceutical composition is administered in the form of a singledosage unit, and can be prepared by any conventional method that isknown in the pharmaceutical field such as methods disclosed inRemington's Pharmaceutical Science (published by Mack PublishingCompany, Easton, Pa., 1970). An injection preparation may optionallycontain as a pharmaceutical carrier a protein derived from plasma suchas albumin, an amino acid such as glycin, or a carbohydrate such asmannitol, and additionally a buffering agent, a solubilizer, or anisotonic agent, etc. can be contained. When the present pharmaceuticalcomposition is in the form of an aqueous solution preparation or alyophilized preparation, it may preferably contain a surfactant such asTween 80 (registered trade mark), Tween 20 (registered trade mark), etc.in order to avoid aggregation. When the present pharmaceuticalcomposition is a composition for parenteral administration other than aninjection preparation, then it may contain distilled water orphysiological saline solution, polyalkylene glycol such as polyethyleneglycol, an oil derived from plant, hydrogenated naphthalene, etc. Forexample, a pharmaceutical composition such as a suppository forintravaginal administration or rectal administration may contain as aconventional excipient polyalkylene glycol, vaseline, cacao butter, etc.A pharmaceutical composition for intravaginal administration may containan absorbefacient such as a bile salt, an ethylenediamine salt, acitrate, etc. A pharmaceutical composition for inhalation may be in theform of a solid preparation, and may contain as an excipient lactose,etc., and a pharmaceutical composition for intranasal drop may be in theform of an aqueous solution or an oily solution.

The present pharmaceutical composition is especially preferable in theform of a formulation by which the present compound can persistently begiven to a subject by a single administration for a long term, e.g., forone week or one year, and various sustained release preparations, depotpreparations, or implant preparations can be employed. For example, apharmaceutical composition may contain a neurotrophic factor per se, ora pharmaceutically acceptable salt of a neurotrophic factor of whichsolubility in body fluid is low. Such pharmaceutically acceptable saltsare, for example, (1): an acid addition salt such as phosphate, sulfate,citrate, tartrate, tannate, pamoate, alginate, polyglutarate,naphthalenemono- or di-sulfonate, polygalacturonate, etc., (2): a saltor complex with polyvalent metal cation such as zinc, calcium, bismuth,barium, nickel, etc, or a combination of (1) and (2), for example, atannic acid zinc salt, etc. A neurotrophic factor is preferablyconverted into a slightly-water-soluble salt thereof, which is mixedwith a gel, for example, aluminum monostearate gel and sesame oil, etc.to give a suitable injection preparation. In this case, especiallypreferable salt is a zinc salt, a tannic acid zinc salt, a pamoate, etc.Another type of a sustained release injection preparation is oneswherein a neurotrophic factor is preferably converted into aslightly-water-soluble salt thereof, which is further enclosed in aslow-disintegrative non-toxic and non-antigenic polymer such as apolymer or a copolymer of polylactic acid/polyglycolic acid. In thiscase, especially preferable salt is zinc salt, tannic acid zinc salt,pamoate, etc. In addition, a neurotrophic factor or aslightly-water-soluble salt thereof can be enclosed into a cholesterolmatrix or collagen matrix to give a sustained release preparation.

The pharmaceutical preparation for oral administration may be ones whichare prepared by microencapsulating a neurotrophic factor or a saltthereof with lecithin, cholesterol, a free fatty acid, or ones which areprepared by enclosing said microcapsules into gelatin capsules, or oneswhich are prepared by enclosing a neurotrophic factor or a salt thereofin enteric capsules, etc. These preparations may additionally contain,for example, an absorbefacient, a stabilizer, a surfactant, etc.

The agent for protecting or ameliorating pancreatic cell function or theagent for protecting or ameliorating pancreatic tissue function can beadministered alone or together with insulin to a patient with pancreaticfunction disorder. The present agents can prevent pancreatichypofunction of a patient with pancreatitis or pancreatic cancer, andcan enable said patient to easily control the blood glucose level ormetabolism. When the present agent is administered together withinsulin, the dosage of insulin is in the range of 4 to 100units/human/day in terms of human insulin, and the daily dosage of aneurotrophic factor, which is administered simultaneously or in advancewith insulin, is in the range of about 1 to about 2500 μg, preferably inthe range of about 10 to about 500 μg per 1 kg of the body weight of anadult.

(Toxicity)

When a neurotrophin, especially BDNF, was administered subcutaneously torats and cynomolgus monkeys at a dose of 100 mg/kg and 60 mg/kg,respectively, for four weeks, no animal died. With respect to the acutetoxicity, BDNF was administered to rats and cynomolgus monkeys at a doseof 200 mg/kg, and no animal died. Therefore, BDNF shows high safety.

The present invention is illustrated by Examples.

EXAMPLE 1 Ameliorating Effects of BDNF on Pancreatic Tissue Picture ofDiabetic Animal Model

(1) Materials and Method for Experiment

Reagents: BDNF was purchased from REGENERON PHARMACEUTICALS, INC. (USA),and used. The other reagents were commercially available ones with thebest quality.

Test animals: Male C57 BL/Ksj-db/db Jcl mice (SPF standard) werepurchased from Clea Japan, Inc., as a diabetic animal model. MaleC57BL/KsJ-db/m Jcl mice (SPF standard) were purchased from Clea Japan,Inc., as a normal mouse. After pre-feeding, the animals were used in theexperiment at 11 weeks old. The mice were kept in a room controlled at atemperature of 23±2° C. under a humidity of 55±10%, with an illuminationcycle of light on (8:00 to 20:00) and light off (20:00 to 8:00). Duringthe pre-feeding, the animals were freely fed with animal chaw (CE-2,Clea Japan, Inc.) and sterilized tap water.

(2) Preparation of BDNF Solution and Administration Thereof

A BDNF solution was prepared by diluting with a phosphate buffer (10 mMphosphoric acid, 150 mM sodium chloride, pH 7.0±0.2) containing 1%mannitol and 0.01% Tween 80 to a concentration of 20 mg/ml, and used inthe experiment. The solution was administered subcutaneously once a dayat a dose of 20 mg/kg/day, for 3 weeks.

(3) Preparation of Pancreatic Extract and Measurement of Insulin Contentand Glucagon Content Therein

After the 3-weeks administration of BDNF, the spleen side of thepancreas was excited, and the weight thereof was measured. The excitedtissue was homogenized in an acid-ethanol, and the mixture was allowedto stand at 4° C. overnight. The mixture was centrifuged, and thesupernatant was kept at −20° C. The insulin concentration in the extractwas assayed by sandwich ELISA (Lewis™ insulin-mouse ELISA kit, ShibayagiCo., Ltd.), and the glucagon concentration in the extract was measuredby competitive RIA (glucagon RIA kit, Rinco Research, Ltd.). Thecontents thereof were calculated from the concentration in the extractand the weight of the tissue. The results are shown in Table 1 and Table2, respectively.

TABLE 1 Insulin content Group (ng/mg of tissue weight) Normal controlmouse 187.7 ± 25.3  Diabetic control mouse + 64.9 ± 21.3 a Vehicleadministration Diabetic mouse + 672.3 ± 130.0 b BDNF administration c

TABLE 2 Glucagon content Group (ng/mg of tissue weight) Normal controlmouse 1.88 ± 0.36 Diabetic control mouse + 6.87 ± 1.61 b Vehicleadministration Diabetic mouse + 4.08 ± 0.44 b BDNF administration c a P< 0.05 vs. Normal control (Turkey's test) b P < 0.01 vs. Normal Control(Turkey's test) c P < 0.01 vs. Diabetic mouse + Vehicle administration(Turkey's test)

(4) Tissue Staining of Pancreas

After the 3-weeks administration of BDNF, the duodena side of thepancreas was excised, and fixed with Bouin's solution, and a paraffinblock was prepared therefrom by a conventional method. From thisparaffin block a section slide of thickness of 3 μm was prepared, and itwas subjected to aldehyde fuchsin staining, insulin immune staining,glucagon immune staining, and somatostatin immune staining.

As a control, the normal mouse and the diabetic mouse were treated, andthe pancreas thereof was subjected to tissue staining in the same manneras above.

As to the stained tissues obtained in the above, a photomicrograph by anoptical microscope was obtained (size bar indicates 200 μm). Thesepictures are shown in FIG. 1. In FIG. 1, A is the stained picture byaldehyde fuchsin staining, B is the stained pictures by insulin immunestaining, C is the stained pictures by glucagon immune staining, and Dis the stained pictures by somatostatin immune staining. The db/db(vehicle) indicates the diabetic control mouse, and the db/db (BDNF)indicates the diabetic BDNF-treated mouse, and the db/m (normal)indicates the normal control mouse.

(5) Results

(i) Amelioration of Insulin and Glucagon Contents in Pancreas by BDNFAdministration

As is apparent from the results as shown in the above Table 1 and Table2, when comparing with the normal control mice, the insulin content inpancreas of the diabetic control mice was reduced to about 25%, whilethe glucagon content in pancreas thereof was about 3 times higher. Onthe contrary, the insulin content in pancreas of the diabetic mice towhich BDNF has been administered for 3 weeks was increased to about 10times as that of the diabetic control mice. On the other hand, theglucagon content in pancreas of said BDNF-treated mice was reduced toabout 50% of that of the diabetic control mice. From these results, itwas confirmed that BDNF can normalize the pancreatic function ofdiabetic mouse by increasing the insulin content in pancreas andreducing the glucagon content in pancreas.

(ii) Histological Amelioration of Pancreatic Tissue by BDNFAdministration

From the results of aldehyde fuchsin staining as shown in FIG. 1, theapparent decrease of insulin granules was observed in the pancreas ofthe diabetic control mouse, as compared with the pancreas of a normalmouse. On the other hand, the distinguished re-granulation of insulingranules was observed in the BDNF-treated group. The similar result wasalso obtained in the stained pictures by insulin immune staining. In theglucagon immune staining and the somatostatin immune staining, theinternalization of glucagon-positive cells and somatostatin-positivecells was observed in the diabetic control mouse. On the other hand,these cells exist in the marginal part of Langerhans islet of theBDNF-treated group, as a similar manner as in the normal control mouse.Thus, it was indicated that the amelioration of pancreatic function byBDNF is mediated not only by the increase in insulin content in insulinpositive cells but also by the normalization of the glucagon positivecell function.

EXAMPLE 2 Examination of Langerhans Islet B Cells by Electron Microscopy

(1) According to the procedure of Example 1, BDNF was administered todb/db mice of 8 weeks old for 6 weeks, and the spleen side of thepancreas thereof was excised and pre-fixed with 2.5% glutaraldehydefixative, and further post-fixed with 2% osmic acid fixative,dehydrated, and embedded in an epoxy resin. The ultrathin section wasstained with uranium acetate and lead citrate, and the Langerhans isletB cells were examined by a transmission electron microscope (JEM 1200EXII, JEOL Ltd.) (magnification: 8300 and 16000). The pictures thereofare shown in FIG. 3 and FIG. 6, respectively.

As a normal control animal, C57 BL/6N mice (SPF standard) were purchasedfrom Charles River Japan Inc., and they were used at 14 weeks old forsampling without any treatment. In the same manner as in the aboveBDNF-treated mice, the Langerhans islet B cells were examined by atransmission electron (magnification: 8300 and 16000). The picturesthereof are shown in FIG. 4 and FIG. 7, respectively. As a diabeticcontrol mouse, the same diabetic mice as used in Example 1 were used,and they were treated in the same manner. The electron micrographs(magnification: 8300 and 16000) are shown in FIG. 2 and FIG. 5,respectively.

(2) Results

As is shown in FIG. 4 and FIG. 7, there were multiple insulin secretorygranules in the cytoplasm of the B cells of the normal control C57BLmice, and it was observed that mitochondria and Golgi apparatus weredisseminated therein, while rough endoplasmic reticulum was scarce andnot clear.

On the other hand, as shown in FIG. 2 and FIG. 5, there was an apparentdecrease in secretory granules in the diabetic control db/db mice. Inaddition, the increase in rough endoplasmic reticulum, the enlargementof Golgi apparatus, and the jumboizing (hypertrophy) of mitochondriawere observed, and they indicated the hypofunction of pancreatic B cellendocrine.

On the contrary, as shown in FIG. 3 and FIG. 6, the reaccumulation ofsecretory granules was observed in the BDNF-treated db/db mice. Inaddition, the increase in rough endoplasmic reticulum, the enlargementof Golgi apparatus, and the jumboizing of mitochondria were hardlyobserved, and the tissue electron micrography thereof was similar tothat of the normal C57BL mice.

From the above results, it was confirmed that the pancreatic endocrinefunction of db/db mice is normalized by BDNF administration.

INDUSTRIAL APPLICABILITY

The present agent comprising as the active ingredient a neurotrophicfactor exhibits an ameliorating effect on pancreatic function disorderbeing induced by various causes, and can protect the pancreatic cells ortissues, and further exhibits an ameliorating effects on thehypofunction of damaged pancreatic cells or tissues, and it is useful asan agent for protecting or ameliorating pancreatic function disorder.

What is claimed is:
 1. A method of protecting pancreatic cell functionor of ameliorating hypofunction of pancreatic cells in a subjectpresenting type II diabetes and a defect in insulin secretion, whichcomprises administering a brain-derived neurotrophic factor to thesubject.
 2. A method of protecting pancreatic tissue function or ofameliorating hypofunction of pancreatic tissues in a subject presentingtype II diabetes and a defect in insulin secretion, which comprisesadministering a brain-derived neurotrophic factor to the subject.
 3. Themethod according to claim 1, wherein the pancreatic cell is selectedfrom the group consisting of B cells (β cells), A cells, (α cells), Dcells (δ cells) of pancreatic Langerhans islet and mixtures thereof. 4.The method according to claim 2, wherein the pancreatic tissue ispancreatic Langerhans islet.
 5. A method of ameliorating pancreaticendocrine function disorder in a subject, which comprises administeringan effective amount of a brain-derived neurotrophic factor to thesubject.
 6. A method of protecting pancreatic cell function or ofameliorating hypofunction of pancreatic cells or tissues in a subjectexhibiting a disorder of pancreatic endocrine or exocrine hormonesecretion, which comprises administering an amount of a brain-derivedneurotrophic factor to the subject effective to protect pancreatic cellfunction or ameliorate hypofunction of pancreatic cells or tissues. 7.The method of claim 6 in which the pancreatic cell hypofunction isameliorated, the hypofunction is hypofunction of islet cells and thesubject exhibits a defect in insulin secretion.
 8. The method of claim6, in which the administration of the brain-derived neurotrophic factorprovides re-granulation of insulin granules.